This is a competing renewal application of R01 AI51622 entitled Chemical Mycobacteriology. Mycobacterium tuberculosis (Mtb) infections are difficult to treat owing to the requirement of multiple drugs administered over many months, the emergence of drug-resistant strains, and a complex lifecycle that can include a drug-refractory latent stage. Mtb adapts to diverse environments during disease progression by influencing host cells and altering its own metabolic state. Glycolipids of the outermost capsular layer are thought to contribute to host-pathogen interactions; their underlying biosynthetic machineries might offer new targets for Mtb therapy. Metabolic pathways essential for survival during latency are also attractive drug targets. The broad objectives of this program are (1) to investigate the functions of mycobacterial cell wall glycolipids, and (2) to explore sulfur metabolism as a new niche for drug discovery. During the last granting period we focused our studies on the Mtb-specific trehalose glycolipid sulfolipid-1 (SL-1), a putative Mtb virulence factor. We elucidated the complete genetic and biosynthetic machinery underlying SL-1 and probed its functions in host cells and animals. In the course of these studies we discovered a novel sulfated menaquinone metabolite in Mtb, termed S881, disruption of which produces a hypervirulent phenotype in the mouse infection model. We also validated ATP sulfurylase, the enzyme catalyzing the first committed step in sulfate assimilation, as an attractive drug target. The next granting period will focus on four specific aims, the first two of which builds directly from previous work. In Aim 1 we will develop small molecule inhibitors of ATP sulfurylase as anti-tuberculosis drugs with possible application toward latent Mtb. In Aim 2 we will investigate the biosynthesis and function of S881, which we propose to play a role in adaptive respiration. We will also initiate two new research directions. In Aim 3 we will study trehalose glycolipids in the Mtb relative Mycobacterium marinum, exploiting the zebrafish infection model to profile their expression during infection. Two emerging technologies, MALDI mass spectrometry imaging and metabolic/bioorthogonal labeling for fluorescence imaging, will be employed to probe the dynamics of trehalose glycolipids during the course of disease. Finally, in Aim 4 we will investigate pathways that modulate cell wall structure in response to osmotic stress. A chemical approach to imaging peptidoglycan in vivo will be developed to facilitate these studies.